Sample collection for microbial analysis
Verification of decontamination is recommended, especially after a disease outbreak or after a food safety incident. This may be performed by a regulatory authority or farm staff. Verification will depend on the circumstances, such as a notifiable disease.
Sterile Whirl-Pak Speci-Sponge environmental surface sampling bags are recommended for microbial testing before and after decontamination. Before sampling, the Whirl-Pak Speci-Sponge must be moistened with 20 ml of sterile buffered peptone water. Wear gloves while sampling and change gloves between different sample types (floors, walls, drinkers, feeders).
For floor and wall samples, each swab should cover an area of approximately 0.5 m2. A minimum of 10 floor and 10 wall samples should be collected from sheds.
A pooled sample can be collected using one swab for 10 drinker cups. A minimum of four such pooled samples must be collected from drinker lines located in various points in the shed. The bottom and insides of drinker cups, and the pipe between two drinker cups, should be swabbed. Care should be taken not to get any water on the swab from the drinker’s nipples, as water will alter the microbial counts for drinker cup swabs.
Pooled samples should be collected using one swab for five feeder pans. In the case of trough feeders, one swab should cover a length of 0.5 meters. The outer sides, bottom and inner sides of the feeder pans/feeding trough should be swabbed.
Other structures that can be swabbed are egg belts, nest pads, nest boxes, dwarf wall and fans (determine a standard size sample for these structures that would cover approximately 0.5 m2). An alternate method for floor sample collection is collecting boot covers (Appendix 6 – Figure 9). Boot cover samples can be collected by walking around inside the shed. After walking inside the shed with boot covers on, remove them and place them in a sterile sealable bag. The method to put on the boot cover for sample collection is detailed in Appendix 6.
Processing of swab samples for total microbial load
Squeeze swabs and transfer the contents into sterile 15 mL tubes. Add 100 μL of the individual sample to 900 μL of 0.9% NaCl, serially diluted, and plate 100 μL onto nutrient agar plates (Thermo Scientific, Australia). Add 20 ml of buffered peptone water to the boot cover samples and massage the sample for one minute. Collect 100 μL of the sample and add to 900 μL of 0.9% NaCl, serially diluted, and plate 100 μL on nutrient agar plates (Thermo Scientific, Australia). Incubate the plates overnight at 37 °C. Determine the total viable count by counting the number of colonies on the nutrient agar plates and calculating the colony forming units (CFU) per m2 of the surface.
For the isolation of Salmonella species, incubate the leftover BPW samples overnight at 37 °C. Next, transfer 100μL of the incubated samples into 10 mL of Rappaport-Vassiliadis soya peptone (RVS) broth (Oxoid, Australia) and incubate at 42 °C for 24 hours. Streak a loop full of the incubated RVS broth onto xylose lysine deoxycholate agar (XLD) (Thermo Scientific, Australia) and incubate at 37 °C for 24 hours. To confirm Salmonella by standard PCR, suspend the suspected Salmonella colony from the XLD agar plates in the nutrient broth (Oxoid, Australia) and incubate at 37 °C in a shaking incubator for 12 hours.
Sample collection for viral verification
For verification of decontamination after a viral disease such as avian influenza, it is best to collect dry samples of various surfaces in the shed. The sample collection should be supervised by government staff. A sterile Flexi swab should be used for viral agent verification. Use the swab-to-swab surfaces such as the wall, drinker cups, feeders, egg belts, fans, nest boxes and the floor. Once the desired areas are swabbed, place the swab in a vial of viral transport media (VTM). Viral transport media contains balanced salt or saline solutions with a buffering capacity to maintain a ‘near-neutral’ pH. This helps stabilise the viruses until the sample can be tested in a lab.
What tests will be performed in the lab will be determined by the government agency involved in responding to the incident. Procedures for handling samples requires development.
A quantitative PCR method was developed to determine the load of infectious bursal disease virus (IBDV) in the shed environment. Note that this exercise was carried out because IBDV is considered one of the hardier viruses affecting the Australian poultry industry, and the unique strains endemic in Australia are avirulent and likely to be present on most chicken farms, so is a good indicator organism when assessing the success of shed decontamination.
